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Chapter 28

Chapter 28. Manipulating DNA. Chapter Objectives. Know how the techniques of molecular biology work Understand how to use the tools of molecular biology Put it all together with a molecular biology project (we will work on this in class after we know the tools and techniques). Techniques.

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Chapter 28

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  1. Chapter 28 Manipulating DNA

  2. Chapter Objectives • Know how the techniques of molecular biology work • Understand how to use the tools of molecular biology • Put it all together with a molecular biology project (we will work on this in class after we know the tools and techniques)

  3. Techniques • Restriction Enzymes • Electrophoresis • Hybridization probes • DNA sequencing • Chain terminator • Automated • Emulsion • PCR

  4. Restriction Enzymes • https://www.neb.com/products/restriction-endonucleases • App: Search NEB tools in either play store or App store • Remember overhangs, this will be important

  5. Electrophoresis Polyacrylamide Agarose

  6. Hybridization Probes • Northern Blot – RNA detection • Southern Blot – DNA detection • Western Blot – Protein detection

  7. DNA Sequencing • Chemical Sequencing • Chain Terminator (dideoxy nucleotides) • Automated (color) • Emulsion

  8. PCR 55? annealing 72 elongate 95 denature repeat

  9. Tools • RT-PCR • Microarray • Cloning • Plasmids • Bacteriaphage • Cosmids • BAC/YAC • Site-directed mutagenesis • Knockout mice

  10. Real-time (kind of) PCR(qPCR) (sort of) • Can quantify the exact about of mRNA using PCR • Combine dsDNA binding dye with PCR

  11. Microarrays

  12. Cloning • Plasmid – small, circular DNA – can hold about 10 kb • Bacteriophage – larger, circular DNA – can hold about 20 kb • Cosmid – combination of a plasmid and phage, can hold 40-50 kb • BAC/YAC – bacterial artificial chromosome/yeast AC – can hold up to 500 kb

  13. Site Directed Mutagenesis • Change DNA in plasmid, etc. to make a mutation in protein (substitution/deletion) • Can change any residue to any other residue • Implications?

  14. Mice • Knockout • Deletion of gene • Knockin • Insertion of gene • Genetic background • models

  15. Active Learning • Projects • Download serial cloner • http://serialbasics.free.fr/Serial_Cloner.html • Build plasmids (learn about functions) • Create (on paper) a mutant protein • Create an experiment to study your protein • Learn about creating knockout mice.

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